Methods and constructs for production of lentiviral vector

Abstract

To provide a method for producing mammalian cells containing a lentiviral packaging vector.SOLUTION: A method of producing a lentiviral packaging vector-containing mammalian cells, comprising: a. transfecting a mammalian cell with a packaging vector comprising an expression cassette encoding: 1. a lentiviral regulator of expression of virion proteins (REV) gene under control of a first promoter; 2. a lentiviral envelope gene under control of a second promoter; and 3. a lentiviral group specific antigen (GAG) gene and a lentiviral polymerase (POL) gene both under control of a third promoter where the expression cassette is flanked on both the 5' and 3' ends by transposon-specific inverted terminal repeats (ITR); b. culturing the transfected mammalian cell; and c. isolating the lentiviral packaging vector-containing mammalian cell.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on September 22, 2020, is named 0132-0051WO1_SL.txt and is 40,945 bytes in size.

[0002] The present disclosure relates to methods for producing lentiviral vector-producing cells. In particular, these methods utilize two, rather than four, plasmids to provide the necessary packaging elements and transfer the vector into cells, allowing for the production of large numbers of lentiviral-producing cells, including suspension-based cells, and the production of large quantities of lentivirus. These methods allow for the production of cells that can later be induced to produce lentivirus and can be tailored to contain specific genes of interest. [Background technology]

[0003] Lentiviral vectors are one of the most commonly used delivery methods in the field of gene and cell therapy. In the lentiviral vector production process, the sequences required for vector production are split across several different plasmids or expression cassettes to minimize the possibility of producing replication-competent lentivirus (RCL). Generally, third-generation lentiviral production systems utilize four separate plasmids or expression cassettes to express: 1) lentivirus group-specific antigen (GAG) genes and lentivirus polymerase (POL) proteins; 2) envelope protein (usually vesicular stomatitis virus glycoprotein (VSV-G)); 3) HIV regulator of virion protein expression (Rev) protein; and 4) Transfer vector (TV) containing the gene of interest (GOI).

[0004] The most common approach involves transiently transfecting cells with the four plasmids mentioned above to generate lentiviral vectors, but this is labor-intensive and expensive. Moreover, transient transfection requires large amounts of plasmid DNA, limiting scalability and raising safety concerns.

[0005] What is needed to overcome the difficulties associated with transient transfection is a method for preparing producer cell lines (PCLs) in which all or most of the genes or sequences required for lentiviral vector production are stably integrated into the chromosomes of mammalian cells that allow lentiviral vector production by simple induction methods. This invention addresses this need. Summary of the Invention

[0006] In some embodiments, provided herein are methods of producing mammalian cells containing a lentiviral packaging vector, the method comprising transfecting mammalian cells with a packaging vector comprising an expression cassette encoding a lentiviral virion protein expression regulator (REV) gene under the control of a first promoter, a lentiviral envelope gene under the control of a second promoter, and a lentiviral group-specific antigen (GAG) gene and a lentiviral polymerase (POL) gene, both under the control of a third promoter, wherein the expression cassette is flanked on both the 5' and 3' ends by transposon-specific inverted terminal repeats (ITRs); culturing the transfected mammalian cells; and isolating the lentiviral packaging vector-containing mammalian cells.

[0007] Also provided herein is a method for producing lentiviral vector-producing mammalian cells, the method comprising: transfecting mammalian cells with a packaging vector containing an expression cassette encoding a lentiviral virion protein expression regulator (REV) gene under the control of a first promoter, a lentiviral envelope gene under the control of a second promoter, and a lentiviral group-specific antigen (GAG) gene and a lentiviral polymerase (POL) gene, both under the control of a third promoter, wherein the expression cassette is flanked on both the 5' and 3' ends by transposon-specific inverted terminal repeats (ITRs); and a transfer vector containing a nucleic acid sequence encoding a gene of interest under the control of a fourth promoter, wherein the nucleic acid sequence is flanked on both the 5' and 3' ends by transposon-specific inverted terminal repeats (ITRs); culturing the transfected mammalian cells; and isolating the lentiviral vector-producing mammalian cells.

[0008] In a further embodiment, provided herein is a method for producing a lentiviral vector, comprising producing a mammalian cell containing a lentiviral packaging vector according to the methods described herein, transfecting the mammalian cell with a transfer vector comprising a nucleic acid sequence encoding a gene of interest under the control of a fourth promoter, inducing production of the expression cassette and nucleic acid, culturing the transfected mammalian cell, and recovering the lentiviral vector.

[0009] Also provided herein is a method of producing a lentiviral vector, comprising producing a lentiviral vector-producing mammalian cell according to the methods described herein, inducing production of an expression cassette and nucleic acid, culturing the mammalian cell, and recovering the lentiviral vector.

[0010] In a further embodiment, provided herein is a method of lentiviral vector therapy comprising administering to a mammalian subject a lentiviral vector produced according to the methods described herein.

[0011] In a further embodiment, provided herein is a mammalian cell that produces a lentiviral vector, the mammalian cell comprising a nucleic acid molecule chromosomally integrated in the mammalian cell, the nucleic acid molecule comprising a lentiviral virion protein expression regulator (REV) gene under the control of a first promoter, a lentiviral envelope gene under the control of a second promoter, and a lentiviral group-specific antigen (GAG) gene and a lentiviral polymerase (POL) gene, both under the control of a third promoter, wherein the nucleic acid sequence is flanked on both the 5' and 3' ends by sequences resulting from transposon-specific inverted terminal repeat (ITR) recombination.

[0012] Also provided herein is a method of producing a lentiviral vector, the method comprising transfecting a mammalian cell described herein with a transfer vector, the transfer vector comprising a nucleic acid sequence encoding a gene of interest under the control of a fourth promoter, inducing production of the expression cassette and nucleic acid, culturing the transfected mammalian cell, and recovering the lentiviral vector.

[0013] In a further embodiment, provided herein is a method of producing a lentiviral vector comprising inducing production of a chromosomally integrated nucleic acid sequence encoding a gene of interest in a mammalian cell as described herein, the chromosomally integrated nucleic acid sequence, culturing the mammalian cell, and recovering the lentiviral vector. [Brief explanation of the drawings]

[0014] [Figure 1]FIG. 1 shows a packaging plasmid according to an embodiment of the present disclosure. [Figure 2] FIG. 2 shows a transfer vector according to an embodiment of the present disclosure. [Figure 3] 3A-3D show HEK293T cells transfected with packaging and transfer vectors according to embodiments herein. [Figure 4] FIG. 4 shows the production of lentiviral vectors using exemplary cell lines described herein. [Figure 5] FIG. 5 shows lentiviral titers for PEI and Lipofectamine transfections with and without transposase. [Figure 6] Figure 6 shows lentiviral productivity and stability of the single cell clone DH4. [Figure 7] Figure 7 shows lentiviral productivity and stability of the single cell clone ED8. [Figure 8] FIG. 8 shows the results of the copy number analysis. [Figure 9] Figure 9 shows a comparison of the potency of lentiviruses produced using transient transfection and producer cell lines. [Figure 10] FIG. 10 shows an exemplary manufacturing flow of antibiotic-free lentiviral PCL during part of a seed train culture. DETAILED DESCRIPTION OF THE INVENTION

[0015] In the claims and / or this specification, the use of the words "a" or "an," when used in conjunction with the term "comprising," may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0016] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the method / device being employed to determine the value. Typically, the term is meant to encompass a variation of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, depending on the context.

[0017] Use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer only to alternatives, or the alternatives are mutually exclusive, but the disclosure supports a definition that refers only to alternatives and "and / or."

[0018] As used in this specification and claims, the terms "comprising" (and including any of its forms such as "comprise" and "comprises"), "having" (and including any of its forms such as "have" and "has"), "including" (and including any of its forms such as "includes" and "include"), or "containing" (and including any of its forms such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited, elements or method steps. It is contemplated that any embodiment discussed herein can be implemented with respect to any method, system, host cell, expression vector, and / or composition of the invention. Furthermore, any of the methods described herein can be achieved using the compositions, systems, cells, and / or nucleic acids of the invention.

[0019] As used herein, "nucleic acid," "nucleic acid molecule," or "oligonucleotide" refers to a polymeric compound containing covalently linked nucleotides. The term "nucleic acid" includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which may be single-stranded or double-stranded. DNA includes, but is not limited to, complementary DNA (cDNA), genomic DNA, plasmid or vector DNA, and synthetic DNA. RNA includes, but is not limited to, mRNA, tRNA, rRNA, snRNA, microRNA, miRNA, or miRNA.

[0020] As used herein, "gene" refers to an assembly of nucleotides that encodes a polypeptide, and includes cDNA and genomic DNA nucleic acid molecules. "Gene" also refers to a nucleic acid fragment that can act as a regulatory sequence preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. In some embodiments, a gene is integrated with multiple copies. In some embodiments, a gene is integrated in a predefined number of copies.

[0021] Lentiviral vectors and their production Lentiviral vectors are a well-studied vector system based on the human immunodeficiency virus (HIV-1). Other lentiviral systems have also been developed as gene transfer systems, including HIV-2, simian immunodeficiency virus, non-primate lentiviruses, feline immunodeficiency virus, and bovine immunodeficiency virus. Driven by safety concerns due to the pathogenicity of HIV-1 in humans, the most widely used lentiviral systems used for clinical and research and development purposes are: 1) Lentivirus group-specific antigen (GAG) genes and lentivirus polymerase (POL) proteins, 2) envelope protein (usually vesicular stomatitis virus glycoprotein (VSV-G)); 3) the HIV regulator of virion protein expression (Rev) protein, and 4) a transfer vector (TV) containing the gene of interest (GOI); It is based on a four-plasmid system expressing

[0022] Traditionally, mammalian cells, such as human embryonic kidney cells (e.g., HEK293), are transfected with each of the four plasmids as adherent cell cultures, and then the desired lentivirus containing the gene of interest is produced. Generally, these transiently transfected cells are capable of producing lentivirus.

[0023] Lentiviral vectors are generally produced with a gene of interest to be introduced into desired cells for therapies and disease treatments, including immunodeficiencies and neurodegenerative diseases.

[0024] The present invention provides improved methods for producing lentivirus, including methods for preparing lentivirus-producing cell lines that can be grown in suspension and that can significantly increase lentivirus production.

[0025] Lentivirus producer cell lines In embodiments, the present specification provides a method for producing mammalian cells containing lentiviral packaging vectors.As used herein, "lentiviral packaging vector-containing cells" refers to cells that contain the elements necessary for producing lentiviral vectors integrated into their genome, but lack the desired gene of interest that should be carried by the lentiviral vector.The lentiviral packaging vector-containing cells can later be transfected with, for example, a transfer vector containing the gene of interest, and then induced to produce the desired lentivirus for the final delivery of the gene of interest.

[0026] Suitably, cells that can be produced using the various methods described herein are mammalian cells and cell lines or cultures. As used herein, the term "mammalian cells" includes cells derived from any member of the mammalian order, such as human cells, mouse cells, rat cells, monkey cells, hamster cells, etc. In some embodiments, the cells are mouse cells, human cells, Chinese hamster ovary (CHO) cells, CHOK1 cells, CHO-DXB11 cells, CHO-DG44 cells, CHOK1SV cells, including all variants (e.g., POTELLIGENT®, Lonza, Slough, UK), and CHOK1SV GS-KO (glutamine synthetase knockout) cells, including all variants (e.g., XCEED™, Lonza, Slough, UK). Exemplary human cells include human embryonic kidney (HEK) cells, such as HEK293, HeLa, or HT1080 cells.

[0027] Mammalian cells include mammalian cell cultures, which can be either adherent or suspension cultures. Adherent cultures refer to cells grown on a substrate surface, such as a plastic plate, dish, or other suitable cell culture growth platform, and can be anchorage-dependent. Suspension cultures refer to cells that can be maintained, for example, in culture flasks or large suspension tanks, which allow for a large surface area for gas and nutrient exchange. Suspension cell cultures often utilize stirring or agitation mechanisms to provide adequate mixing. Media and conditions for maintaining cells in suspension are generally known in the art. An exemplary suspension cell culture includes human HEK293 clonal cells.

[0028] In embodiments, the methods described herein include transfecting mammalian cells with a packaging vector containing an expression cassette. As used herein, a "vector" or "expression vector" is a replicon, such as a plasmid, phage, virus, or cosmid, to which a nucleic acid molecule described herein can be attached to effect replication and / or expression of the attached nucleic acid molecule in the cell. "Vector" includes episomal vectors (e.g., plasmids) and non-episomal vectors. The term "vector" includes both viral and non-viral means for introducing a nucleic acid molecule into a cell in vitro, in vivo, or ex vivo. The term vector can include synthetic vectors. Vectors can be introduced into desired host cells by known methods, including, but not limited to, transfection, transduction, cell fusion, and lipofection. Vectors can include various regulatory elements, including promoters.

[0029] As used herein, "transfection" refers to the introduction of an exogenous nucleic acid molecule, including a vector, into a cell. A "transfected" cell contains an exogenous nucleic acid molecule within the cell, and a "transformed" cell is one in which the exogenous nucleic acid molecule within the cell induces a phenotypic change in the cell. The transfected nucleic acid molecule can be integrated into the host cell's genomic DNA and / or can be maintained extrachromosomally by the cell, either transiently or long-term. A host cell or organism that expresses an exogenous nucleic acid molecule or fragment is referred to as a "recombinant," "transformed," or "transgenic" organism. Many transfection techniques are commonly known in the art. See, for example, Graham et al., Virology, 52:456 (1973); Sambrook et al., Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York (1989); Davis et al., Basic Methods in Molecular Biology, Elsevier (1986); and Chu et al., Gene 13:197 (1981). Suitably, transfection of mammalian cells with one or more of the vectors described herein utilizes a transfection agent such as polyethyleneimine (PEI) or other suitable agent, including various lipids and polymers, to integrate the nucleic acid into the genomic DNA of the host cell.

[0030] As used herein, "packaging vector" refers to a vector that contains the components necessary to produce a lentiviral vector and "packages" a gene of interest into the final lentivirus. Packaging vectors include an expression cassette, which refers to a separate component of a vector, and contains one or more genes and regulatory sequences that are inserted into and ultimately expressed by transfected cells.

[0031] Suitably, the expression cassette used in the packaging vector comprises a lentiviral virion protein expression regulator (REV) gene under the control of a first promoter, a lentiviral envelope gene under the control of a second promoter, and a lentiviral group-specific antigen (GAG) gene and a lentiviral polymerase (POL) gene, both under the control of a third promoter. In other embodiments, a single promoter can control expression of each of the REV, ENV, GAG, and POL genes, or one promoter can control expression of GAG and POL, and a second promoter controls expression of REV and ENV. Other combinations are possible and encompassed herein.

[0032] The lentiviral virion protein regulator of expression (REV) is an RNA-binding protein that promotes late gene expression and is also important for the transport of unspliced ​​and singly spliced ​​mRNAs encoding viral structural proteins from the nucleus to the cytoplasm.

[0033] The lentiviral envelope (ENV) gene, suitably the vesicular stomatitis virus glycoprotein (VSV-G) gene, encodes a polyprotein precursor that is cleaved by cellular proteases into the surface (SU) envelope glycoprotein gp120 and the transmembrane (TM) glycoprotein gp41.

[0034] GAG encodes a polyprotein that is translated from unspliced ​​mRNA and cleaved by the viral protease (PR) into matrix, capsid, and nucleocapsid proteins. Lentiviral polymerase (POL) is expressed as the GAG-POL polyprotein as a result of ribosomal frameshifting during GAG mRNA translation and encodes the enzyme proteins reverse transcriptase, protease, and integrase. These three proteins are associated with the viral genome within the virion. Appropriately, the GAG ​​gene is the HIV GAG gene, and the POL gene is the HIV POL gene.

[0035] In a preferred embodiment, the expression cassette is flanked on both the 5' and 3' ends by transposon-specific inverted terminal repeats (ITRs). As described in detail herein, it is the use of transposon ITRs (in combination with the corresponding transposase) that allows for the expression cassette to be specifically integrated into the genome of the target cell.

[0036] The method for producing mammalian cells containing a lentiviral packaging vector further includes culturing the transfected mammalian cells to allow the desired nucleic acid (i.e., the expression cassette) to be integrated into the genome of the cells, and then isolating the mammalian cells containing the lentiviral packaging vector.

[0037] Methods for culturing transfected mammalian cells are known in the art and include the use of various cell culture media, appropriate gas concentration / exchange and temperature control to promote cell growth and integration of the construct into the cell's genome.

[0038] Methods for isolating the desired cells include a variety of filtration techniques including sieves, filter devices, cell selectors and sorting, cell counting, and the like.

[0039] As described herein, each component of the expression cassette is under the control of a promoter. As used herein, "under control" refers to a gene controlled by a "promoter," "promoter sequence," or "promoter region," which refers to a DNA regulatory region / sequence capable of binding RNA polymerase and initiating transcription of a downstream coding or non-coding gene sequence. In other words, the promoter and gene are in operable combination or operably linked. As referred to herein, the terms "in operable combination," "in operable order," and "operably linked" refer to the linking of nucleic acid sequences in such a way as to produce a promoter capable of directing the transcription of a given gene and / or the synthesis of a desired protein molecule. The terms also refer to the linking of amino acid sequences in such a way that a functional protein is produced.

[0040] In some examples of the present disclosure, the promoter sequence includes a transcription initiation site and extends upstream to include the minimum number of bases or elements necessary to initiate transcription at a level detectable above background. In some embodiments, the promoter sequence includes a transcription initiation site as well as a protein binding domain involved in the binding of RNA polymerase. Eukaryotic promoters often, but not always, contain "TATA" and "CAT" boxes. A variety of promoters, including inducible promoters, can be used to drive gene expression, for example, in the host cells or vectors of the present disclosure. In some embodiments, the promoter is not a leaky promoter, i.e., the promoter does not constitutively express any of the gene products described herein. In other embodiments described herein, the promoter is a constitutive promoter that initiates mRNA synthesis independent of external regulatory influences.

[0041] Suitably, the promoters used to control the transcription of the various genes in the expression cassette are derepressible promoters. As used herein, a "derepressible promoter" refers to a structure comprising a functional promoter and an additional element or sequence that can bind to a repressor element to cause repression of the functional promoter. "Repression" refers to the reduction or inhibition of transcription initiation of a downstream coding or non-coding gene sequence by the promoter. A "repressor element" refers to a protein or polypeptide that can bind to a promoter (or to the vicinity of a promoter) to reduce or inhibit the promoter's activity. A repressor element can interact with a substrate or binding partner of the repressor element, resulting in a conformational change in the repressor element. This conformational change in the repressor element removes the ability of the repressor element to reduce or inhibit the promoter, resulting in "derepression" of the promoter, thereby allowing the promoter to proceed with transcription initiation. A "functional promoter" refers to a promoter that lacks the action of a repressor element and would still be able to initiate transcription. A variety of functional promoters that can be used in the practice of the present invention are known in the art and include, for example, promoters of PCMV, PH1, P19, P5, P40, and adenovirus helper genes (e.g., E1A, E1B, E2A, E4Orf6, and VA).

[0042] Exemplary repressor elements and their corresponding binding partners that can be used as derepressible promoters are known in the art, and include the cumate gene switch system (CuO operator, CymR repressor, and cumate binding partner) (see, e.g., Mullick et al., "The cumate gene-switch: a system for regulated expression in mammalian cells," BMC Biotechnology 6:43 (1-18) (2006), the disclosure of which is incorporated herein by reference in its entirety, including the disclosure of the derepressible promoter system described herein) and the TetO / TetR system described herein (see, e.g., Yao et al., "Tetracycline Repressor, tetR, rather than the tetR-Mammalian Cell Transcription Factor Fusion Derivatives, Regulates Inducible Gene Expression in Mammalian Cells," Human GeneTherapy 9:1939-1950). (1998), the disclosure of which is incorporated herein by reference in its entirety. In exemplary embodiments, the derepressible promoter comprises a functional promoter and one of two tetracycline operator sequences (TetO or TetO2). Suitably, the expression cassette further encodes repressor elements of the first, second, and third derepressible promoters, including a tetracycline repressor protein.

[0043] A schematic diagram illustrating a derepressible promoter system is provided in Figure 1, which depicts an exemplary packaging plasmid according to an embodiment of the present invention. Of note is a derepressible promoter comprising a CMV promoter containing a TetO sequence (CMV-TO). The CMV-TO derepressible promoter is illustrated operably linked to the REV, VSV-G, and GAG-POL genes; i.e., the first, second, and third promoters can all be derepressible promoters in preferred embodiments. Also depicted is another promoter system, the TetR repressor element under the control of the hPGK promoter.

[0044] When the tetracycline repressor protein (the repressor element of the TetR-TetO sequence) binds to the TetO sequence, CMV promoters are repressed; that is, little or no transcription occurs from these promoters. Upon binding of a binding partner for TetR (appropriately, doxycycline (Dox)), the TetR protein changes conformation, releasing it from the TetO sequence, and the functional promoter begins normal transcription processing as in the native promoter.

[0045] As shown in Figure 1, the expression cassette can further include a Kruppel-associated box (KRAB) sequence following the sequence encoding a repressor element, suitably a tetracycline repressor protein. The KRAB sequence (approximately 75 amino acids) is a transcriptional repression domain from human zinc finger protein 10, which provides increased regulation of the repressor element, suitably a TetR repressor element. The KRAB domain functions as a transcriptional repressor when tethered to template DNA by the DNA-binding domain. Also shown are exemplary locations of 5' and 3' ITR transposon sequences. As previously mentioned, the ITR sequences are positioned to allow the entire expression cassette to be transposed into the target cell, thereby allowing all of the desired genes to be inserted into the host genome.

[0046] In exemplary embodiments, the expression cassette further comprises one or more reporter genes for determining proper integration of the cassette into the cell's genome. As referred to herein, a "reporter gene" is a gene whose expression confers a phenotype on a cell that can be easily identified and measured. In some embodiments, a reporter gene comprises a fluorescent protein gene. In some embodiments, a reporter gene comprises a selection gene. As referred to herein, the term "selection gene" refers to the use of a gene encoding an enzymatic activity that confers the ability to grow in a medium lacking what may be an essential nutrient. In addition, a selection gene can confer resistance to antibiotics or drugs on cells in which the selection gene is expressed. A selection gene can be used to confer a specific phenotype on a host cell. When a host cell must express a selection gene to grow in a selective medium, the gene is called a positive selection gene. A selection gene can also be used to select against host cells containing a specific gene; a selection gene used in this manner is called a negative selection gene. In exemplary embodiments, the selection gene is located downstream of the KRAB sequence following the repressor protein and also downstream of an internal ribosome entry site (IRES) sequence. In exemplary embodiments, the selection gene is an antibiotic resistance gene, including, for example, genes that confer resistance to gentamicin, thymidine kinase, ampicillin, purinycin, and / or kanamycin.

[0047] As described in detail herein, the use of transposon-specific ITRs has been determined to allow for the insertion of expression cassettes into the genome of target cells with increased specificity, frequency, and stability. In an exemplary embodiment, the transposon-specific ITRs are Lepidoptera transposon (PIGGYBAC®) ITRs.

[0048] Transposable elements (transposons) can move within the genome of a cell and are useful for inserting genes to produce transgenic organisms. The lepidopteran transposon PIGGYBAC® can move within the genomes of a wide variety of organisms and is useful as a gene transfer vector. The transposon structure contains a complex repeat sequence consisting of internal repeats (IRs), spacers, and terminal repeats (TRs) at both ends, and a single open reading frame encoding a transposase.

[0049] The lepidopteran transposable element PIGGYBAC® was originally isolated from TN-368 nettle looper cell cultures as a gene disrupting insertions in naturally occurring baculovirus plaque-forming mutants. PIGGYBAC® is a 2475-bp short inverted repeat with an asymmetric terminal repeat structure, with a 3-bp spacer between the 5' 13-bp TR (terminal repeat) and 19-bp IR (internal repeat), and a 31-bp spacer between the 3' TR and IR. A single 2.1-kb open reading frame encodes a functional transposase (Cary et al., 1989; Fraser et al., 1983, 1995; Elick et al., 1996a; Lobo et al., 1999; Handler et al., 1998). PIGGYBAC® transposes via a unique cut-and-paste mechanism, inserting only into the 5′TTAA3′ target site that is duplicated upon insertion, and excising precisely without leaving a footprint (Elick et al., 1996b; Fraser et al., 1996; Wang and Fraser 1993).

[0050] Exemplary lepidopteran transposon (PIGGYBAC®) ITRs that can be used in the plasmids and expression cassettes described herein include those disclosed in U.S. Pat. No. 7,105,343, the disclosure of which is incorporated herein by reference in its entirety.

[0051] In a preferred embodiment, transfection of mammalian cells with the packaging vector is carried out in the presence of a transposase that recognizes the transposon-specific ITRs. The transposase facilitates transposition of the expression cassette into the cellular genome of the target mammalian cell. The transposase can be provided to the cell as an active enzyme or as a nucleic acid sequence encoding the transposase, including mRNA or cDNA. In an embodiment, the transposase is a Lepidoptera (PIGGYBAC®) transposase mRNA or a Lepidoptera (PIGGYBAC®) transposase cDNA. As described herein, the frequency of transposition utilizing the Lepidoptera (PIGGYBAC®) transposon ITRs and the corresponding Lepidoptera (PIGGYBAC®) transposase is suitably at least about 10 -4 is.

[0052] In a further embodiment, the present invention provides a method for producing lentiviral vector-producing mammalian cells. As used herein, "lentiviral vector-producing cells" refers to cells that contain the elements necessary for producing lentiviral vectors integrated into their genome, as well as the desired gene of interest carried by the lentiviral vector. The lentiviral vector-producing cells can then be induced to produce the desired lentivirus for the ultimate delivery of the gene of interest.

[0053] A method for producing lentiviral vector-producing cells, particularly mammalian cells, involves transfecting mammalian cells with a packaging vector containing an expression cassette encoding a lentiviral virion protein expression regulator (REV) gene under the control of a first promoter, a lentiviral envelope gene under the control of a second promoter, and a lentiviral group-specific antigen (GAG) gene and a lentiviral polymerase (POL) gene, both under the control of a third promoter.

[0054] As described herein, the expression cassette is suitably flanked on both the 5' and 3' ends by transposon-specific inverted terminal repeats (ITRs) to facilitate high transposition into target cells.

[0055] The method further includes transfecting the mammalian cell with a transfer vector containing a nucleic acid sequence encoding a gene of interest under the control of a fourth promoter. Like the expression cassette, the nucleic acid sequence encoding the gene of interest is suitably flanked on both the 5' and 3' ends by transposon-specific inverted terminal repeats (ITRs) to enhance transposition. In some embodiments, both the transfer vector and packaging vector are transfected simultaneously; in other embodiments, the transfer vector or packaging vector can be transfected first, followed by the other vector.

[0056] After transfection, the mammalian cells are cultured and lentiviral vector-producing mammalian cells are isolated.

[0057] As with the lentiviral packaging vector-containing cells, the lentiviral vector-producing cells prepared herein are appropriately stored prior to the desired time when they can be used to produce lentiviral vectors. Suitable storage techniques and characteristics are known in the art and can include refrigeration or freezing of the cells, as well as other methods of maintaining the cells in suspension prior to induction of vector production.

[0058] As described herein, suitably the mammalian cells are mammalian cell cultures, in embodiments suspension cultures. Example cells include HEK293T cells.

[0059] As in the case of the lentiviral packaging vector-containing cell, the genes of the expression vector suitably include the GAG ​​gene, which is the HIV GAG gene, and the POL gene, which is the HIV POL gene. Suitably, the lentiviral envelope gene is the vesicular stomatitis virus glycoprotein (VSV-G) gene.

[0060] Exemplary promoters for use in lentiviral vector production cells are known in the art and include derepressible promoters, and suitably the expression cassette further encodes a repressor element for the first, second, and third derepressible promoters. In embodiments, the derepressible promoter comprises a functional promoter and a tetracycline operator sequence (TetO), and the repressor element is a tetracycline repressor protein. Further features of expression cassettes comprising transposon-specific ITRs are described herein.

[0061] In a suitable embodiment, the gene of interest contained in the lentiviral vector is a gene of therapeutic interest. As referred to herein, the term "gene of interest" or "GOI" is used to describe a heterologous gene. As referred to herein, the term "heterologous gene" or "HG" refers to a nucleic acid sequence, such as a gene, that is associated with a nucleic acid sequence, such as a coding sequence or a regulatory sequence, and is not normally linked together and / or is not normally associated with a specific cell. In some embodiments, a heterologous gene is a construct in which the coding sequence itself is not found in nature (e.g., a synthetic sequence with codons different from those of a natural gene). Allelic variations or naturally occurring mutation events do not result in heterologous DNA as used herein.

[0062] As referred to herein, the term "therapeutic gene" refers to any functionally related nucleotide sequence. Thus, therapeutic genes of the present disclosure can include any desired gene encoding a protein defective or missing in the therapeutic target cell genome, or encoding a non-native protein with a desired biological or therapeutic effect (e.g., antiviral function), or the sequence can correspond to a molecule with antisense or ribozyme function. Representative (non-limiting) examples of suitable therapeutic genes include those used to treat inflammatory, autoimmune, chronic, and infectious diseases, including disorders such as AIDS, cancer, neurological disorders, cardiovascular disease, and hypercholesterolemia; various blood disorders, including various anemias, thalassemias, and hemophilias; and genetic defects such as cystic fibrosis, Gaucher disease, adenosine deaminase (ADA) deficiency, and emphysema. Several antisense oligonucleotides (e.g., short oligonucleotides complementary to sequences surrounding the translation start site (AUG codon) of mRNA) that are useful in antisense therapy for cancer and viral diseases have been described in the art, as well as examples of suitable genes for therapeutic purposes.

[0063] Figure 2 shows an exemplary transfer vector containing a gene of interest (enhanced green fluorescent protein, for illustrative purposes). The gene of interest is under the control of a promoter, suitably a constitutive promoter, in this case the CMV promoter. Also shown are exemplary locations of 5' and 3' transposon sequences. As previously mentioned, the ITR sequences are positioned to allow the entire nucleic acid, including the gene of interest and promoter, to be transferred to the target cell, allowing all of the desired gene to be inserted into the host genome. As shown, the exemplary transfer vector also contains a selection gene, in this case a bleomycin resistance gene downstream of the gene of interest. Additional elements of the transfer vector are shown in Figure 2.

[0064] In a further embodiment, the present disclosure provides a method for producing a lentiviral vector, comprising producing or providing mammalian cells containing the lentiviral packaging vector described herein, and then transfecting the mammalian cells with a transfer vector comprising a nucleic acid sequence encoding a gene of interest under the control of a fourth promoter. Following the introduction of the gene of interest into the cell genome, the production of the nucleic acid encoding the expression cassette and the gene of interest is induced. The cells are then cultured, and finally, the lentiviral vector containing the gene of interest is recovered.

[0065] In yet a further embodiment, a method for producing a lentiviral vector preferably comprises producing or providing a mammalian cell that produces a lentiviral vector as described herein. The production of an expression cassette and a nucleic acid encoding a gene of interest is induced in the cell. The cell is then cultured, and finally, the lentiviral vector containing the gene of interest is recovered.

[0066] The methods of producing lentiviral vectors using cells described herein, whether they contain simply a lentiviral packaging vector or also a gene of interest integrated into the cellular genome, provide a mechanism for producing large quantities of lentiviral vector as well as controlling when induction begins and the conditions under which induction occurs. Induction suitably involves the introduction of a chemical or agent that interacts with the repressor element, thereby derepressing the derepressible promoter and allowing production of the packaging components of the lentiviral vector.

[0067] As described herein, each promoter in the expression cassette is suitably a derepressible promoter comprising a functional promoter and a tetracycline operator sequence (TetO), and the repressor element is a tetracycline repressor protein. In such embodiments, induction of the packaging components comprises adding doxycycline to the mammalian cell.

[0068] The methods described herein provide increased production of lentiviral vectors as a result of increased insertion of desired nucleic acid sequences into the cellular genome, and suspension-based cell cultures that allow for large-scale production. In embodiments, the amount of lentiviral vector produced is at least about 10 4 , or more appropriately, about 10 5 or about 10 6 transducing units / mL within about 1 to 5 days, suitably about 2 days after induction of the cells.

[0069] The cell-based production methods described herein can utilize any suitable reactor, including, but not limited to, stirred tank, airlift, fiber, microfiber, hollow fiber, ceramic matrix, fluidized bed, fixed bed, and / or spouted bed bioreactors. As used herein, a "reactor" can include a fermentor or fermentation unit or any other reaction vessel, and the term "reactor" is used interchangeably with "fermentor." The term fermentor or fermentation refers to both microbial and mammalian cultures. For example, in some embodiments, an exemplary bioreactor unit can perform one or more or all of the following: feeding nutrients and / or carbon sources, injecting a suitable gas (e.g., oxygen), flowing fermentation or cell culture media in and out, separating gas and liquid phases, maintaining temperature, maintaining oxygen and CO2 concentrations, maintaining pH concentration, agitating (e.g., stirring), and / or cleaning / sanitizing. An example reactor unit, such as a fermentation unit, can include multiple reactors within the unit; for example, a unit can have 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more bioreactors within each unit, and / or a facility can include multiple units with single or multiple reactors within the facility. In various embodiments, the bioreactors can be suitable for batch, semi-fed-batch, fed-batch, perfusion, and / or continuous fermentation processes. Any suitable reactor diameter can be used. In embodiments, the bioreactors can have a volume between about 100 mL and about 50,000 L.Non-limiting examples include 100 mL, 250 mL, 500 mL, 750 mL, 1 liter, 2 liters, 3 liters, 4 liters, 5 liters, 6 liters, 7 liters, 8 liters, 9 liters, 10 liters, 15 liters, 20 liters, 25 liters, 30 liters, 40 liters, 50 liters, 60 liters, 70 liters, 80 liters, 90 liters, 100 liters, 150 liters, 200 liters, 250 liters, 300 liters, 350 liters, 400 liters, 450 liters, 500 liters, and 550 liters. 10,000 liters, 15,000 liters, 20,000 liters, and / or 50,000 liters. Additionally, suitable reactors may be multi-use, single-use, disposable, or non-disposable and may be formed from any suitable material, including stainless steel (e.g., 316L or other suitable stainless steel) and metal alloys such as Inconel, plastic, and / or glass.

[0070] It has surprisingly been determined that at least a portion of the method for producing lentivirus using producing cell lines as described herein can be performed in the absence of antibiotics, including seed train production, cell passaging, large-scale cell culture, and / or key manufacturing steps. By eliminating antibiotics from at least a portion of the lentivirus production process, the resulting method reduces or eliminates concerns associated with the use of antibiotics in cell processing and ultimately in products for human use.

[0071] Figure 10 shows an exemplary manufacturing process flow for preparing lentivirus from a producer cell line as described herein. As shown, PCL clones are initially generated and then selected for use in a large-scale manufacturing process. During the early stages of the manufacturing process, PCL are passaged approximately three times (e.g., to a volume of approximately 2 L), at which point antibiotics such as puromycin and / or zeocin can be removed from the process. Passaging can then be continued in the absence of antibiotics, e.g., at passage 3, passage 4, passage 5, etc., until a desired volume of cells is reached. For example, as shown in Figure 10, a volume of approximately 50 L is suitably reached in a bioreactor in the absence of antibiotics. The PCL are then suitably induced (e.g., using doxycycline as described herein) to begin production of lentiviral vectors. Thus, in embodiments, at least a portion of the culture of the mammalian cells (PCL) occurs in the absence of antibiotics, suitably from passage 3 through the induction phase. After induction, downstream processing can occur, including recovery (including benzonase treatment and clarification steps) and lentiviral vector purification (including tangential flow filtration and sterile filtration). The ability to perform a significant portion of the lentiviral vector manufacturing process using PCL as described herein is a surprising and unexpected result, and offers significant advantages for large-scale manufacturing processes.

[0072] Also provided herein are methods of treating mammalian subjects, suitably human subjects, with lentiviral vectors produced according to the various methods described herein. Suitably, the methods are used to treat human subjects with a gene of interest, including a gene of therapeutic interest. Administration to a human subject can include, for example, inhalation, injection, or intravenous administration, as well as other administration methods known in the art.

[0073] Also provided herein are mammalian cells for producing lentiviral vectors. Using the methods described herein, or variations thereof, mammalian cells can be readily produced that contain a chromosomally integrated nucleic acid molecule, the nucleic acid molecule comprising a lentiviral virion protein expression regulator (REV) gene under the control of a first promoter, a lentiviral envelope gene under the control of a second promoter, and a lentiviral group-specific antigen (GAG) gene and a lentiviral polymerase (POL) gene, both under the control of a third promoter. In a further embodiment, the mammalian cell containing the genomically integrated packaging components can further comprise a chromosomally integrated nucleic acid sequence encoding a gene of interest under the control of a fourth promoter.

[0074] As described herein, using transposase-based methods, including the PIGGYBAC® transposase, nucleic acid sequences in a host cell genome become flanked on both the 5' and 3' ends by sequences resulting from transposon-specific inverted terminal repeat (ITR) recombination.

[0075] Exemplary mammalian cells are described herein, as are the genetic components of the expression cassette and nucleic acid encoding the gene of interest.

[0076] Methods for utilizing these cells to produce lentivirus are described herein and suitably include inducing production of an expression cassette and nucleic acid encoding a gene of interest, culturing the transfected mammalian cells, and recovering the lentiviral vector.

[0077] Further exemplary embodiments Embodiment 1 is a method of producing mammalian cells containing a lentiviral packaging vector, comprising transfecting mammalian cells with a packaging vector comprising an expression cassette encoding a lentiviral virion protein expression regulator (REV) gene under the control of a first promoter, a lentiviral envelope gene under the control of a second promoter, and a lentiviral group-specific antigen (GAG) gene and a lentiviral polymerase (POL) gene, both under the control of a third promoter, wherein the expression cassette is flanked on both the 5' and 3' ends by transposon-specific inverted terminal repeats (ITRs); culturing the transfected mammalian cells; and isolating the lentiviral packaging vector-containing mammalian cells.

[0078] Embodiment 2 includes the method of embodiment 1, wherein the mammalian cells are a mammalian cell culture.

[0079] Embodiment 3 includes the method of embodiment 2, wherein the mammalian cell culture is a suspension culture.

[0080] Embodiment 4 includes the method of Embodiment 3, wherein the mammalian cells are HEK293T cells.

[0081] Embodiment 5 includes the method of any one of embodiments 1 to 4, wherein the GAG ​​gene is an HIV GAG gene and the POL gene is an HIV POL gene.

[0082] Embodiment 6 includes the method of any one of Embodiments 1 to 5, wherein the lentiviral envelope gene is a vesicular stomatitis virus glycoprotein (VSV-G) gene.

[0083] Embodiment 7 includes the method of any one of embodiments 1 to 6, wherein the first, second, and third promoters are derepressible promoters.

[0084] Embodiment 8 includes the method of embodiment 7, wherein the expression cassette further encodes repressor elements for the first, second, and third derepressible promoters.

[0085] Embodiment 9 includes the method of embodiment 8, wherein each of the derepressible promoters comprises a functional promoter and a tetracycline operator sequence (TetO), and the repressor element is a tetracycline repressor protein.

[0086] Embodiment 10 includes the method of embodiment 9, wherein the expression cassette further comprises a Kruppel-associated box sequence following the sequence encoding the tetracycline repressor protein.

[0087] Embodiment 11 includes the method of any one of Embodiments 1 to 10, wherein the transposon-specific ITRs are Lepidoptera transposon (PIGGYBAC®) ITRs.

[0088] Embodiment 12 includes the method of any one of embodiments 1 to 11, wherein the transfection is in the presence of a transposase that recognizes the transposon-specific ITRs.

[0089] Embodiment 13 includes the method of embodiment 12, wherein the transposase is Lepidoptera (PIGGYBAC®) transposase mRNA or Lepidoptera (PIGGYBAC®) transpose cDNA.

[0090] Embodiment 14 is a method for producing lentiviral vector-producing mammalian cells, comprising transfecting mammalian cells with a packaging vector comprising an expression cassette encoding a lentiviral virion protein expression regulator (REV) gene under the control of a first promoter, a lentiviral envelope gene under the control of a second promoter, and a lentiviral group-specific antigen (GAG) gene and a lentiviral polymerase (POL) gene, both under the control of a third promoter, wherein the expression cassette is flanked on both the 5' and 3' ends by transposon-specific inverted terminal repeats (ITRs); and a transfer vector comprising a nucleic acid sequence encoding a gene of interest under the control of a fourth promoter, wherein the nucleic acid sequence is flanked on both the 5' and 3' ends by transposon-specific inverted terminal repeats (ITRs); culturing the transfected mammalian cells; and isolating the lentiviral vector-producing mammalian cells.

[0091] Embodiment 15 includes the method of embodiment 14, wherein the mammalian cells are a mammalian cell culture.

[0092] Embodiment 16 includes the method of embodiment 15, wherein the mammalian cell culture is a suspension culture.

[0093] Embodiment 17 includes the method of embodiment 16, wherein the mammalian cells are HEK293T cells.

[0094] Embodiment 18 includes the method of any one of embodiments 14 to 17, wherein the GAG ​​gene is an HIV GAG gene and the POL gene is an HIV POL gene.

[0095] Embodiment 19 includes the method of any one of embodiments 14 to 18, wherein the lentiviral envelope gene is the vesicular stomatitis virus glycoprotein (VSV-G) gene.

[0096] Embodiment 20 includes the method of any one of embodiments 14 to 19, wherein the first, second and third promoters are derepressible promoters.

[0097] Embodiment 21 includes the method of embodiment 20, wherein the expression cassette further encodes repressor elements for the first, second, and third derepressible promoters.

[0098] Embodiment 22 includes the method of embodiment 21, wherein each of the derepressible promoters comprises a functional promoter and a tetracycline operator sequence (TetO), and the repressor element is a tetracycline repressor protein.

[0099] Embodiment 23 includes the method of embodiment 22, wherein the expression cassette further comprises a Kruppel-associated box sequence following the sequence encoding the tetracycline repressor protein.

[0100] Embodiment 24 includes the method of any one of embodiments 14 to 23, wherein the transposon-specific ITRs are Lepidoptera transposon (PIGGYBAC®) ITRs.

[0101] Embodiment 25 includes the method of any one of embodiments 14 to 24, wherein the transfection is in the presence of a transposase that recognizes the transposon-specific ITRs.

[0102] Embodiment 26 includes the method of embodiment 25, wherein the transposase is Lepidoptera (PIGGYBAC®) transposase mRNA or Lepidoptera (PIGGYBAC®) transpose cDNA.

[0103] Embodiment 27 includes the method of any one of embodiments 14 to 26, wherein the gene of interest is a gene of therapeutic interest.

[0104] Embodiment 28 is a method for producing a lentiviral vector, the method comprising: producing mammalian cells containing the lentiviral packaging vector described in embodiment 1; transfecting the mammalian cells with a transfer vector, the transfer vector comprising a nucleic acid sequence encoding a gene of interest under the control of a fourth promoter, to induce production of an expression cassette and the nucleic acid; culturing the transfected mammalian cells; and recovering the lentiviral vector.

[0105] Embodiment 29 is a method for producing a lentiviral vector, comprising producing a lentiviral vector-producing mammalian cell as described in embodiment 14, inducing production of an expression cassette and nucleic acid, culturing the mammalian cell, and recovering the lentiviral vector.

[0106] Embodiment 30 includes the method of embodiment 28 or embodiment 29, wherein each of the promoters in the expression cassette is a derepressible promoter comprising a functional promoter and a tetracycline operator sequence (TetO), the repressor element is a tetracycline repressor protein, and the induction comprises adding doxycycline to the mammalian cell.

[0107] Embodiment 31 is a method for producing a lentiviral vector comprising the steps of: 6 30. The method of embodiment 28 or embodiment 29, wherein the amount of transduction units / mL is transducing units / mL.

[0108] Embodiment 32 is a method of lentiviral vector therapy, comprising administering to a mammalian subject a lentiviral vector produced according to embodiment 28 or embodiment 29.

[0109] Embodiment 33 includes the method of embodiment 32, wherein administering comprises inhalation, injection, or intravenous administration.

[0110] Embodiment 34 is a mammalian cell for producing a lentiviral vector, the mammalian cell comprising a nucleic acid molecule integrated into a chromosome, the nucleic acid molecule comprising a lentiviral virion protein expression regulator (REV) gene under the control of a first promoter, a lentiviral envelope gene under the control of a second promoter, and a lentiviral group-specific antigen (GAG) gene and a lentiviral polymerase (POL) gene, both under the control of a third promoter, wherein the nucleic acid sequence is flanked on both the 5' and 3' ends by sequences resulting from transposon-specific inverted terminal repeat (ITR) recombination.

[0111] Embodiment 35 comprises the mammalian cell of embodiment 34, further comprising a chromosomally integrated nucleic acid sequence encoding a gene of interest under the control of a fourth promoter.

[0112] Embodiment 36 includes the mammalian cell of embodiment 34 or embodiment 35, wherein the mammalian cell is a mammalian cell culture.

[0113] Embodiment 37 comprises the mammalian cell of embodiment 36, wherein the mammalian cell culture is a suspension culture.

[0114] Embodiment 38 includes the mammalian cell of embodiment 37, wherein the mammalian cell is a HEK293T cell.

[0115] Embodiment 39 comprises the mammalian cell of any one of embodiments 34 to 38, wherein the GAG ​​gene is an HIV GAG gene and the POL gene is an HIV POL gene.

[0116] Embodiment 40 comprises the mammalian cell of any one of embodiments 34 to 39, wherein the lentiviral envelope gene is the vesicular stomatitis virus glycoprotein (VSV-G) gene.

[0117] Embodiment 41 comprises the mammalian cell of any one of embodiments 34 to 40, wherein the first, second and third promoters are derepressible promoters.

[0118] Embodiment 42 comprises the mammalian cell of embodiment 41, wherein the expression cassette further encodes a repressor element for the first, second, and third derepressible promoters.

[0119] Embodiment 43 comprises the mammalian cell of embodiment 42, wherein each of the derepressible promoters comprises a functional promoter and a tetracycline operator sequence (TetO), and the repressor element is a tetracycline repressor protein.

[0120] Embodiment 44 includes the mammalian cell of embodiment 43, wherein the expression cassette further includes a Kruppel-associated box sequence following the sequence encoding the tetracycline repressor protein.

[0121] Embodiment 45 comprises the mammalian cell of any one of embodiments 34 to 44, wherein the transposon-specific ITRs are Lepidoptera transposon (PIGGYBAC®) ITRs.

[0122] Embodiment 46 comprises the mammalian cell of embodiment 35, wherein the gene of interest is a gene of therapeutic interest.

[0123] Embodiment 47 is a method for producing a lentiviral vector, comprising transfecting a mammalian cell of embodiment 34 with a transfer vector, the transfer vector comprising a nucleic acid sequence encoding a gene of interest under the control of a fourth promoter; inducing production of the expression cassette and nucleic acid; culturing the transfected mammalian cell; and recovering the lentiviral vector.

[0124] Embodiment 48 is a method for producing a lentiviral vector, comprising inducing production of a chromosomally integrated nucleic acid sequence encoding a gene of interest in a mammal of embodiment 35 and the chromosomally integrated nucleic acid sequence, culturing mammalian cells, and recovering the lentiviral vector.

[0125] Embodiment 49 includes the method of embodiment 47 or embodiment 48, wherein each of the promoters in the expression cassette is a derepressible promoter comprising a functional promoter and a tetracycline operator sequence (TetO), the repressor element is a tetracycline repressor protein, and the induction comprises adding doxycycline to the mammalian cell.

[0126] Embodiment 50 is a method for producing a lentiviral vector comprising administering to a subject the method of claim 1, wherein the amount of lentiviral vector produced is at least about 10% by weight two days after induction. 6 49. The method of claim 47 or 48, wherein the amount of transducing units / mL is 1000 mg / mL.

[0127] Embodiment 51 is a method of treating with a lentiviral vector, comprising administering to a mammalian subject a lentiviral vector produced according to embodiment 47 or embodiment 48.

[0128] Embodiment 52 includes the method of embodiment 51, wherein administering comprises inhalation, injection, or intravenous administration.

[0129] Embodiment 53 includes the method of any of embodiments 28-31 and 47-50, wherein at least a portion of the culturing of the mammalian cells occurs in the absence of antibiotics.

[0130] Embodiment 54 includes the method of embodiment 53, wherein the portion of the culture is from passage 3 to the induction phase. Example

[0131] Example 1: Design and construction of lentiviral producer cell lines material and method To construct lentiviral producer cell lines, two plasmids were designed: 1) a packaging plasmid expressing GAG-Pol, VSV-G, and Rev in a regulated manner; and 2) A transfer vector that expresses the gene of interest.

[0132] The coding sequences of VSV-G and Rev have been codon-optimized (co), as this has proven to be one of the most effective ways to increase protein synthesis without changing the actual amino acid sequence of the protein.

[0133] The GAG-Pol coding sequence was not codon-optimized. All of these sequences were placed under the control of the CMV-TO promoter, and their activity was repressed by TetR in the absence of tetracycline or doxycycline. In our design, to achieve tighter regulation of CMV-TO, we inserted the Kruppel-associated box (KRAB) sequence, a transcriptional repression domain from human zinc finger protein 10, immediately after the TetR coding sequence.

[0134] Upon binding to TetR, the addition of doxycycline induces a conformational change in TetR, releasing it from CMV-TO. As a result, CMV-TO becomes activated and expresses VSV-G, Rev, and GAG-Pol. Regulated expression of VSV-G, Rev, and GAG-Pol minimizes any cytotoxicity associated with these proteins and maintains lentiviral vector production at a basal level before any induction. To facilitate selection, an antibiotic resistance marker (puromycin) was introduced into this plasmid downstream of the IRES sequence, which was placed immediately after the KRAB sequence. Thus, the human phosphoglycerate promoter fully drives expression of TetR, KRAB, and the puromycin resistance gene.

[0135] To test lentiviral vector production, we selected enhanced green fluorescent protein (eGFP) as the gene of interest (GOI), which was constitutively expressed under the CMV promoter. To make the selection process more efficient, we introduced a bleomycin resistance marker (BleoR) into the transfer vector.

[0136] In conventional approaches, integration of the expression cassette relies on random integration events. Therefore, it is inherently an inefficient and controlled process. Furthermore, because fragmented partial antibiotic marker(s) with incomplete expression cassettes may accidentally integrate into the chromosome, the final selected clones may be false positives (i.e., do not express the intended GOI).

[0137] To avoid these undesirable events in producer cell line generation, all expression cassettes in the packaging and transfer vector plasmids were flanked by PIGGYBAC® inverted terminal repeat (ITR) sequences. The PIGGYBAC® transposase specifically recognizes these ITR sequences and promotes site-specific recombination in vivo. Thus, upon co-transfection of PIGGYBAC® transposase mRNA, the plasmid region containing the entire expression cassette is efficiently integrated into the cellular chromosome.

[0138] A schematic diagram of the packaging vector is shown in Figure 1, and a schematic diagram of the transfer vector is shown in Figure 2. The promoter-containing eGFP coding sequence is shown. The PIGGYBAC® 5' and 3' ITRs are also shown. Elements for lentiviral vector production, such as the 5' LTR, HIV-1 ψ, RRE, cPPT / CTS, and 3' LTR, are also shown.

[0139] The nucleic acid sequences for the packaging vectors shown in Figure 1 are provided below:

[0140] The nucleic acid sequence for the transfer vector shown in Figure 2 is shown below:

[0141] Single-cell clones of HEK293T cells were isolated and adapted for transfection with packaging and transfer vector plasmids. To test and confirm the effectiveness of the PIGGYBAC® transposase, transfections were performed in the absence or presence of PIGGYBAC® transposase mRNA. PEIpro transfection agent was used for transfection.

[0142] To generate lenti-eGFP packaging cell lines, the packaging plasmid and a transfer vector encoding eGFP were co-transfected. To generate packaging cell lines for packaging alone (i.e., without expression of the gene of interest), the packaging plasmid alone was transfected. Four days after transfection, stably transfected cells were selected by adding puromycin and zeocin for lenti-eGFP and puromycin alone for packaging cell lines.

[0143] result Cotransfection of transposase mRNA significantly enhanced antibiotic-resistant colonies, demonstrating that transposase expression indeed promotes chromosomal integration of the expression cassette. Figures 3A-3D show the results of HEK293T cells transfected with the packaging and eGFP transfer vector in the absence (Figure 3A and Figure 3C) or presence (Figure 3B and Figure 3D) of transposase mRNA (TP mRNA). Four days after transfection, antibiotic-resistant cells were selected by adding puromycin (0.5 μg / mL) and zeocin (300 μg / mL). At 26 days posttransfection, cells were observed under a fluorescent microscope, and GFP and black-and-white (B / W) images were collected.

[0144] Similarly, cotransfection of transposase mRNA enhanced antibiotic-resistant colonies during packaging cell line generation (data now shown).

[0145] Antibiotic-resistant packaging cell lines were amplified and frozen prior to characterization. Packaging cells encoding LV-eGFP were thawed and cultured in suspension in the presence of puromycin and zeocin. To induce lentiviral vector production, sodium butyrate (6 mM) and doxycycline (2 μg / mL) were added to the packaging cell culture and further incubated.

[0146] On days 2 and 3 after induction, culture supernatants were collected and lentiviral titers were measured. Culture supernatants from uninduced samples were used to measure uninduced lentiviral vector titers. At the time of induction (days 2 and 3), more than 4E6 lentiviral vector transduction units / mL were produced, whereas the basal amount of lentiviral vector production before either induction (day 0) was observed to be at a basal level (Figure 4).

[0147] Example 2: Further characterization of lentivirus producer cell lines A. Further studies were designed to explore the use of different transfection agents with the vectors described herein. As shown in Figure 5, both PEI and Lipofectamine 3000 successfully increased infectious lentiviral titers from the PCL pool by approximately 100-fold in the presence of PIGGYBAC® transposase mRNA, demonstrating that expression of the transposase leads to efficient integration of the cargo sequence into the host cell chromosome.

[0148] B. Further experiments were performed to determine the effect of antibiotic removal on successful lentiviral production. For these experiments, a single cell clone (SCC), HEK-293T, designated DH4, was selected because it showed consistent infectious titers upon induction up to passage 21.

[0149] As shown in Figure 6, uninduced cells exhibited low levels of infectious titers. After passage 7 (day 2), induced cells exhibited 1.2E7 TU / mL (transducing units / mL). For cell selection, the antibiotic was removed and cells continued to be passaged. At passage 11 (days 2 and 3), antibiotic-treated cells achieved titers of 1.5E7 TU / mL (day 2) and 5.3E7 TU / mL (day 3). Compared to non-antibiotic treatment, the infectious titer reached 1.6E7 TU / mL even on day 2. Upon expansion to passage 17 and passage 21 (approximately 7 weeks), viral titers continued to maintain similar levels (4.4E7) without antibiotics as with antibiotics. Day 2 vs. day 3 refers to the number of days of incubation of PCLs after induction before harvest, comparing the effect of additional days between induction and harvest.

[0150] Figure 7 shows similar results using another single-cell clone, ED8. As with the DH4 clone, removal of antibiotics at passage 7 did not significantly affect the cell clone's ability to produce infectious virus titers on the same scale as antibiotic-treated cells. As previously noted, by passage 21, an infectious titer of 3.5E7TU / mL was achieved, similar to 4.3E7TU / mL in cells that continued antibiotic treatment.

[0151] Figure 8 shows the copy number (of gene of interest or packaging plasmid) integrated into the host cell chromosome, as measured using a ddPCR assay. The vector copy number (VCN) was calculated using the formula: VCN = copy number of target sequence / copy number of RPP30 (ribonuclease P / MRP subunit P30) * 2. Primer / probe sets specific for the long terminal repeat (LTR) and vesicular stomatitis virus glycoprotein G (VSV-G) were used to detect the GOI and packaging plasmid, respectively. As noted for both cell clones, both the gene of interest and packaging plasmid were efficiently integrated into the cell chromosome. Removal of antibiotics during cell passaging (again at passage 70) resulted in a slight decrease in the copy number, but sufficient integration of the GOI and packaging plasmid into the cells was still observed.

[0152] This is a surprising and unexpected result, demonstrating that even in the absence of antibiotics, producer cell lines prepared according to the methods described herein are capable of producing high viral titers, which provides a significant advantage in lentiviral manufacturing, as it is highly desirable to remove antibiotics from producer cell line cultures to minimize risks associated with antibiotic contamination of products subsequently used in human patient populations.

[0153] Experiments were also performed to determine the transduction efficiency of lentiviral vectors produced using the producer cell lines described herein compared to lentivirus produced from transient transfection. Lentivirus was produced using PCL from both DH4 and ED8 cell lines, along with transient transfection of HEK-293 cells. After production, lentiviral vectors were transduced into human peripheral blood mononuclear cells (PBMCs) at a multiplicity of infection (MOI) of 5. Briefly, PBMCs were plated at 4E5 cells / well in 24-well plates. On day 1, cells were stimulated with IL-2 (15 ng / mL) and anti-CD3 and anti-CD28 (25 μL / 1E6 cells). Next, cells were transduced with lentivirus at a multiplicity of infection of 5 using green fluorescent protein as the gene of interest (GOI). Five days later, cells were analyzed using flow cytometry to determine the fraction of fluorescent-positive cells and calculate transduction efficiency.

[0154] As shown in Figure 9, when PMBCs are transduced at a multiplicity of infection of 5, similar transduction efficiencies are observed for lentivirus produced using transient transfection methods compared to PC1C (DH4 and ED8 lines) and lentivirus produced using the methods described herein (transduction efficiencies of approximately 55%-62%).

[0155] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations to the methods and applications described herein can be made without departing from the scope of any of the embodiments.

[0156] Although specific embodiments have been illustrated and described herein, it is to be understood that the claims are not limited to the specific forms or arrangements of parts described and illustrated. Although exemplary embodiments are disclosed herein, and specific terms are employed, they are used in a generic and descriptive sense only, and not for purposes of limitation. Modifications and variations of the embodiments are possible in light of the above teachings. It is therefore to be understood that the embodiments may be practiced otherwise than as specifically described.

[0157] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. 1. A method for producing mammalian cells containing lentiviral packaging vectors, comprising: a. mammalian cells, i. a packaging vector comprising an expression cassette, 1. a lentiviral virion protein expression regulator (REV) gene under the control of a first promoter; 2. a lentiviral envelope gene under the control of a second promoter, and 3. Encoding a lentiviral group-specific antigen (GAG) gene and a lentiviral polymerase (POL) gene, both under the control of a third promoter; transfecting a packaging vector, wherein the expression cassette is flanked at both the 5' and 3' ends by transposon-specific inverted terminal repeats (ITRs); b. Culturing the transfected mammalian cells; c. isolating mammalian cells containing the lentiviral packaging vector.

2. The method of claim 1 , wherein the mammalian cells are in mammalian cell culture.

3. 3. The method of claim 2, wherein the mammalian cell culture is a suspension culture.

4. The method of claim 3, wherein the mammalian cells are HEK293T cells.

5. The method according to any one of claims 1 to 4, wherein the GAG ​​gene is the HIV GAG gene and the POL gene is the HIV POL gene.

6. The method according to any one of claims 1 to 5, wherein the lentiviral envelope gene is a vesicular stomatitis virus glycoprotein (VSV-G) gene.

7. The method of any one of claims 1 to 6, wherein the first, second and third promoters are derepressible promoters.

8. 8. The method of claim 7, wherein the expression cassette further encodes repressor elements for the first, second and third derepressible promoters.

9. 9. The method of claim 8, wherein each of the derepressible promoters comprises a functional promoter and a tetracycline operator sequence (TetO), and the repressor element is a tetracycline repressor protein.

10. 10. The method of claim 9, wherein the expression cassette further comprises a Kruppel-associated box sequence following the sequence encoding the tetracycline repressor protein.

11. The method of any one of claims 1 to 10, wherein the transposon-specific ITRs are Lepidoptera transposon (PIGGYBAC®) ITRs.

12. The method of any one of claims 1 to 11, wherein the transfecting is in the presence of a transposase that recognizes the transposon-specific ITR.

13. 13. The method of claim 12, wherein the transposase is Lepidoptera (PIGGYBAC®) transposase mRNA or Lepidoptera (PIGGYBAC®) transpose cDNA.

14. 1. A method for producing a lentiviral vector-producing mammalian cell, comprising: a. mammalian cells, i. a packaging vector comprising an expression cassette, 1. a lentiviral virion protein expression regulator (REV) gene under the control of a first promoter; 2. a lentiviral envelope gene under the control of a second promoter, and 3. Encoding a lentiviral group-specific antigen (GAG) gene and a lentiviral polymerase (POL) gene, both under the control of a third promoter; a packaging vector, wherein the expression cassette is flanked on both the 5' and 3' ends by transposon-specific inverted terminal repeats (ITRs); and ii. A transfer vector, 1. A nucleic acid sequence encoding a gene of interest under the control of a fourth promoter; transfecting a transfer vector, wherein the nucleic acid sequence is flanked at both the 5' and 3' ends by transposon-specific inverted terminal repeats (ITRs); b. Culturing the transfected mammalian cells; and c. isolating said lentiviral vector-producing mammalian cells.

15. 15. The method of claim 14, wherein the mammalian cells are in mammalian cell culture.

16. 16. The method of claim 15, wherein the mammalian cell culture is a suspension culture.

17. 17. The method of claim 16, wherein the mammalian cells are HEK293T cells.

18. The method according to any one of claims 14 to 17, wherein the GAG ​​gene is the HIV GAG gene and the POL gene is the HIV POL gene.

19. The method according to any one of claims 14 to 18, wherein the lentiviral envelope gene is the vesicular stomatitis virus glycoprotein (VSV-G) gene.

20. 20. The method of any one of claims 14 to 19, wherein the first, second and third promoters are derepressible promoters.

21. 21. The method of claim 20, wherein the expression cassette further encodes repressor elements for the first, second and third derepressible promoters.

22. 22. The method of claim 21, wherein each of the derepressible promoters comprises a functional promoter and a tetracycline operator sequence (TetO), and the repressor element is a tetracycline repressor protein.

23. 23. The method of claim 22, wherein the expression cassette further comprises a Kruppel-associated box sequence following the sequence encoding the tetracycline repressor protein.

24. 24. The method of any one of claims 14 to 23, wherein the transposon-specific ITRs are Lepidoptera transposon (PIGGYBAC®) ITRs.

25. The method of any one of claims 14 to 24, wherein the transfecting is in the presence of a transposase that recognizes the transposon-specific ITR.

26. 26. The method of claim 25, wherein the transposase is Lepidoptera (PIGGYBAC®) transposase mRNA or Lepidoptera (PIGGYBAC®) transpose cDNA.

27. The method according to any one of claims 14 to 26, wherein the gene of interest is a gene of therapeutic interest.

28. 1. A method for producing a lentiviral vector, comprising: a. Producing a mammalian cell containing the lentiviral packaging vector of claim 1; b. the mammalian cell i. a nucleic acid sequence encoding a gene of interest under the control of a fourth promoter transfecting the cells with a transfer vector comprising: c. Inducing production of the expression cassette and the nucleic acid; d. Culturing the transfected mammalian cells; e. recovering the lentiviral vector.

29. 1. A method for producing a lentiviral vector, comprising: a. Producing a lentiviral vector-producing mammalian cell according to claim 14; b. Inducing production of the expression cassette and the nucleic acid; c. Culturing the mammalian cells; and d. recovering the lentiviral vector.

30. 30. The method of claim 28 or claim 29, wherein each of the promoters in the expression cassette is a derepressible promoter comprising a functional promoter and a tetracycline operator sequence (TetO), the repressor element is a tetracycline repressor protein, and the induction comprises adding doxycycline to the mammalian cell.

31. The amount of the lentiviral vector produced is at least about 10% two days after the induction. ６ 30. The method of claim 28 or claim 29, wherein the amount is transducing units / mL.

32. 1. A method of treating with a lentiviral vector, comprising: a) administering to a mammalian subject the produced lentiviral vector of claim 28 or claim 29.

33. 33. The method of claim 32, wherein said administering comprises inhalation, injection, or intravenous administration.

34. A mammalian cell for producing a lentiviral vector, comprising: a. a nucleic acid molecule integrated into a chromosome in said mammalian cell, said nucleic acid molecule comprising: i. a lentiviral virion protein expression regulator (REV) gene under the control of a first promoter; ii. a lentiviral envelope gene under the control of a second promoter; and iii. a lentiviral group-specific antigen (GAG) gene and a lentiviral polymerase (POL) gene, both under the control of a third promoter; A mammalian cell comprising a nucleic acid molecule, wherein the nucleic acid sequence is flanked on both the 5' and 3' ends by sequences resulting from transposon-specific inverted terminal repeat (ITR) recombination.

35. 35. The mammalian cell of claim 34, further comprising a chromosomally integrated nucleic acid sequence encoding a gene of interest under the control of a fourth promoter.

36. 36. The mammalian cell of claim 34 or claim 35, wherein the mammalian cell is a mammalian cell culture.

37. 37. The mammalian cell of claim 36, wherein the mammalian cell culture is a suspension culture.

38. 38. The mammalian cell of claim 37, wherein the mammalian cell is a HEK293T cell.

39. The mammalian cell according to any one of claims 34 to 38, wherein the GAG ​​gene is an HIV GAG gene and the POL gene is an HIV POL gene.

40. The mammalian cell according to any one of claims 34 to 39, wherein the lentiviral envelope gene is a vesicular stomatitis virus glycoprotein (VSV-G) gene.

41. 41. The mammalian cell of any one of claims 34 to 40, wherein the first, second and third promoters are derepressible promoters.

42. 42. The mammalian cell of claim 41, wherein said expression cassette further encodes repressor elements of said first, second and third derepressible promoters.

43. 43. The mammalian cell of claim 42, wherein each of the derepressible promoters comprises a functional promoter and a tetracycline operator sequence (TetO), and the repressor element is a tetracycline repressor protein.

44. 44. The mammalian cell of claim 43, wherein the expression cassette further comprises a Kruppel-associated box sequence following the sequence encoding the tetracycline repressor protein.

45. 45. The mammalian cell of any one of claims 34 to 44, wherein the transposon-specific ITRs are Lepidoptera transposon (PIGGYBAC®) ITRs.

46. 36. The mammalian cell of claim 35, wherein the gene of interest is a gene of therapeutic interest.

47. 1. A method for producing a lentiviral vector, comprising: a. the mammalian cell of claim 34; i. a nucleic acid sequence encoding a gene of interest under the control of a fourth promoter transfecting the cells with a transfer vector comprising: b. Inducing production of the expression cassette and the nucleic acid; c. Culturing the transfected mammalian cells; and d. recovering the lentiviral vector.

48. 1. A method for producing a lentiviral vector, comprising: a) inducing production of said chromosomally integrated nucleic acid sequence and said chromosomally integrated nucleic acid sequence encoding a mammalian gene of interest of claim 35; b. Culturing the mammalian cells; c. recovering the lentiviral vector.

49. 49. The method of claim 47 or claim 48, wherein each of the promoters in the expression cassette is a derepressible promoter comprising a functional promoter and a tetracycline operator sequence (TetO), the repressor element is a tetracycline repressor protein, and the induction comprises adding doxycycline to the mammalian cell.

50. The amount of the lentiviral vector produced is at least about 10% two days after the induction. ６ 49. The method of claim 47 or claim 48, wherein the concentration is transducing units / mL.

51. 1. A method of treating with a lentiviral vector, comprising: a) administering to a mammalian subject the produced lentiviral vector of claim 47 or claim 48.

52. 52. The method of claim 51, wherein said administering comprises inhalation, injection, or intravenous administration.

53. 51. The method of any one of claims 28 to 31 and 47 to 50, wherein at least a portion of the culturing of the mammalian cells is carried out in the absence of antibiotics.

54. 54. The method of claim 53, wherein the portion of the culture is from passage 3 through the induction phase.

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